The Role Of E3 Ubiquitin Ligase TRIM38 In Cardiac Fibrosis After Myocardial Infarction

Background Cardiac fibrosis following myocardial infarction is a complicated pathophysiological process,which involving multiple molecular and signalling pathways and may lead to cardiovascular events including heart failure and sudden cardiac death.At present,however,the understanding of the molecular mechanism of cardiac fibrosis is limited.TRIM family proteins play an important role in regulating biological processes such as cell proliferation and apoptosis,innate immunity and inflammatory responses.Multiple members of family have been found to be involved in the pathophysiological process of cardiac remodeling.TRIM38,as one of the TRIM family proteins,has typical RING domain,B-box and coiled-coil domains.This structure determines that TRIM38 has E3 ubiquitin ligase activity,which can regulate the ubiquitination of itself or different substrates,and participate in a multitude of complex molecular and cellular mechanisms.This study aimed to clarify the effect and specific mechanism of TRIM38 on cardiac fibrosis.Part Ⅰ Expression changes of TRIM38 in the pathophysiological process of cardiac fibrosisObjective: To explore expression changes of TRIM38 in pathophysiological process of cardiac fibrosis.Methods: Coronary blood samples were collected from patients with acute myocardial infarction,and expression of TRIM38 was detected by enzyme-linked immunosorbent assay(ELISA).The C57BL/6 mice and cardiac fibroblasts from Sprague-Dawley suckling rat were used in this section.The left anterior descending coronary artery ligation procedure or angiotensin II(Ang II)was performed to establish in vivo and in vitro cardiac fibrosis model,respectively.The cardiac function was measured by echocardiography 4 weeks after myocardial infarction.Immunohistochemical staining and Masson staining was performed to evaluate the extent of fibrosis.Western blot was used to detect the protein level of TRIM38 in mouse heart tissue after myocardial infarction.The rat cardiac fibroblasts were isolated and cultured in vitro.Western blot was used to detect the changes of TRIM38 protein expression in cardiac fibroblasts at each time point.Results: The expression of TRIM38 was down-regulated in coronary blood of patients with acute myocardial infarction.The levels of myocardial fibrosis markers α-SMA and Collagen I were increased in the in vivo and in vitro models of myocardial fibrosis.Compared with sham group,the protein expression of TRIM38 in cardiac tissue from the myocardial infarction mice was decreased.In an Ang II-induced myocardial fibrosis model,the protein level of TRIM38 in cardiac fibroblasts decreased in a timedependent manner.Conclusions: TRIM38 was significantly downregulated in myocardial fibrosis induced by myocardial infarction,suggesting that TRIM38 plays an important role in cardiac remodeling.Part Ⅱ The effect of TRIM38 on cardiac fibrosis induced by myocardial infarctionObjective: To investigate effect of TRIM38 on cardiac fibrosis induced by myocardialinfarction.Methods: Adeno-associated virus was used to knockdown or overexpression TRIM38 expression in mice.Then,the myocardial infarction model was produced by ligating the left anterior descending coronary artery.The cardiac function was measured by echocardiography 4 weeks after myocardial infarction.Real-time PCR was used to detect RNA expression of fibrotic markers.HE staining was performed to observe cardiac tissue morphology.The levels of collagen deposition were determined using immunohistochemical staining.Masson staining was performed to evaluate myocardial fibrosis.Results: In response to sham operation,there are no differences in cardiac function,fibrosis-related gene expression levels,and extent of myocardial fibrosis in each group of mice.Echocardiography was conducted 4 weeks after MI.In the AAV9-sh RNATRIM38/MI group,the degree of damage to cardiac function induced by myocardial infarction was more severe than that in the AAV9-sh RNA-NC/MI group,while it was significantly improved in the AAV9-TRIM38/MI group.pathological analysis showed that compared with the AAV9-sh RNA-NC/MI group,the AAV9-sh RNA-TRIM38/MI group had aggravated myocardial fibrosis and increased left ventricular collagen volume fraction,which were effectively alleviated in the AAV9-TRIM38/MI group.Furthermore,PCR assays revealed that m RNA levels of myocardial fibrosis markers Acta2 and Col1α1 in AAV9-sh RNA-TRIM38/MI group were significantly higher than AAV9-sh RNA-NC/MI group,while those in AAV9-TRIM38/MI group were significantly higher lower than the AAV9-NC/MI group.Conclusions: TRIM38 is involved in cardiac remodeling by regulating cardiac fibrotic.Part Ⅲ Effect of TRIM38 on activation of cardiac fibroblasts in vitroObjective: Elucidating the effect of TRIM38 on cardiac fibroblast activation in vitro.Methods: The AdshTRIM38 or AdTRIM38 adenoviruses were used to knockdown or upregulate TRIM38 expression in cardiac fibroblasts,respectively.Cardiac fibroblasts infected with Adsh RNA or Ad GFP were used as controls.The in vitro myocardial fibrosis model was established by Ang II stimulation.After challenge with Ang II or PBS for 48 hours,fibrosis markers were tested by Western blot,immunofluorescence staining and real-time PCR.Cardiac fibroblasts migration was assessed using wound healing assays.Results: In response to PBS administration,the protein and m RNA expression levels of fibroblast markers α-SMA and Collagen I were comparable between Adsh TRIM38,Adsh RNA,Ad TRIM38 and Ad GFP group.48 hours after Ang II treatment,the protein and mRNA expression levels of fibroblast markers α-SMA and collagen I,determined by Western blot and real-time PCR,was increased in Adsh TRIM38 group but decreased in Ad TRIM38 group compared with their control groups,respectively.Additionally,compared with their respective controls,Ang II-induced cardiac fibroblast activation were exaggerated by Adsh TRIM38 but alleviated by Ad TRIM38.The wound healing assay showed that TRIM38 knockdown promoted the proliferation and migration of cardiac fibroblasts,while TRIM38 overexpression inhibited the proliferation and migration of cardiac fibroblasts.Conclusions: TRIM38 is involved in cardiac fibrosis by regulating cardiac fibroblast function.Part Ⅳ The mechanism of TRIM38 regulating cardiac fibrosis after myocardial infarction through TAK1/MAPK signaling axisObjective: Elucidating the molecular mechanism of TRIM38 regulating cardiac fibrosis after myocardial infarction through TAK1/MAPK signaling axis.Methods: In both in vivo and in vitro experiments,Western blot was used to detect the protein levels of phosphorylated and total TAK1/MAPK signaling pathway molecules in each group.Then,the TRIM38 knockdown cardiac fibroblasts were stimulated with Ang II to construct an in vitro fibrosis model,and the TAK1-specific inhibitor 5Z-7-Oxozeaenol was used to treat cardiac fibroblasts,and relevant indicators were detected 48 hours later.The protein levels of phosphorylated and total TAK1/MAPK signaling pathway molecules were detected by Western blot.Cardiac fibroblast activation was assessed by immunohistochemical staining.Results: The total expression of TAK1/MAPK signaling molecules was not altered by pro-fibrotic stimulus,whereas the phosphorylated levels of TAK1/MAPK signaling molecules was elevated in fibrosis model.Among them,the phosphorylated levels of TAK1/MAPK signaling molecules was further elevated by TRIM38 overexpression but alleviated by TRIM38 deficiency.In an in vitro cardiac fibrosis model induced by Ang II,the TAK1-specific inhibitor 5Z-7-Oxozeaenol treatment could markedly decrease the phosphorylation level of TAK1/MAPK in TRIM38 knockdown cardiac fibroblasts and inhibit cardiac fibroblasts activation.Conclusions: TRIM38 is involved in cardiac fibrosis by inhibiting the activation of TAK1/MAPK signaling pathway.Part Ⅴ TRIM38 inhibits TAK1 activation by regulating the degradation of TAB2/3Objective: To elucidate the molecular mechanism by which TRIM38 regulates TAB2/3 degradation and inhibits TAK1 activation.Methods: The experiments were performed with cardiac fibroblasts isolated from neonatal Sprague-Dawley rats.Recombinant adenoviruses for TRIM38 silencing or overexpression were transfected into cardiac fibroblasts.Then,the in vitro myocardial fibrosis model was established by Ang II stimulation.The effect of TRIM38 on the ubiquitination level of TAK1 and the interaction between TRIM38 and TAB2/3 was ascertained by co-immunoprecipitation experiments.Effect of TRIM38 on the stability of TAB2/3 protein was ascertained by western blot.HA-TAB2/3 and Flag-TRIM38 plasmids were transfected in HEK293 T cells.By performing cell immunofluorescence,the colocalization of TRIM38 and TAB2/3 was identified under a laser confocal microscope;the interaction between TRIM38 and TAB2/3 was identified by coimmunoprecipitation experiment.Gradient transfection of HA-TAB2/3 and FlagTRIM38 in HEK293 T cells to ascertain the effect of TRIM38 on the stability of TAB2/3 protein;and addition of DMSO,MG132,and CQ,respectively,to ascertain approach of TRIM38 affecting stability of TAB2/3 protein.The effects of TRIM38 on TAB2/3-mediated activation of TAK1 ubiquitination and phosphorylation in HEK293 T cells were ascertained.Results: TRIM38 inhibited the Lys63 ubiquitination activation of TAK1;TRIM38 interacted with TAB2/3,respectively,and degraded TAB2/3 via lysosomal pathway.TRIM27 inhibited TAB2/3-mediated ubiquitination and phosphorylation activation of TAK1.Conclusions: TRIM38 inhibits the activation of TAK1 signaling pathway by degrading TAB2/3 and subsequently regulates cardiac fibrosis.